Synthetic lethal interactions between EGFR and PARP inhibition in human triple negative breast cancer cells.

Few therapeutic options exist for the highly aggressive triple negative breast cancers (TNBCs). In this study, we report that a contextual synthetic lethality can be achieved both in vitro and in vivo with combined EGFR and PARP inhibition with lapatinib and ABT-888, respectively. The mechanism involves a transient DNA double strand break repair deficit induced by lapatinib and subsequent activation of the intrinsic pathway of apoptosis. Further dissection of the mechanism reveals that EGFR and BRCA1 can be found in the same protein complex, which is reduced by lapatinib. Interestingly, lapatinib also increases cytosolic BRCA1 and EGFR, away from their nuclear DNA repair substrates. Taken together, these results reveal a novel regulation of homologous recombination repair involving EGFR and BRCA1 interaction and alteration of subcellular localization. Additionally, a contextual synthetic lethality may exist between combined EGFR and PARP inhibitors

Lapatinib attenuates homologous recombination mediated DNA double strand break repair in triple negative breast cancer cells.

<p>(A–C) Homologous recombination (HR) repair capacity was measured in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 triple negative breast cancer cell lines by assessing radiation-induced rad51 foci, a well characterized marker for HR repair. Briefly, cells were exposed to mock or 3 Gy irradiation (IR) and subsequently subjected to immunofluorescence staining for rad51 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with rad51 foci (**p<0.01 compared to vehicle). (D) Chromosomal HR repair capacity was directly measured in MDA-MB-231DRGFP cells. MDA-MB-231DRGFP were treated with 1 µM lapatinib or vehicle control. 16 hours following the treatment period, cells were transfected with ISce-1 or control vector. 48 hours following transfection cells were subjected to flow cytometry for GFP expression. Shown is the representative fold induction in GFP (mean +/− SEM) from at least 3 independent experiments (**p<0.01 compared to vehicle). Inset is a representative figure depicting the DRGFP repair model.</p

Treatment with lapatinib sequesters EGFR to the cytosol in triple negative breast cancer cells.

<p>(A–C) Cells were subjected to 1 µM lapatinib treatment for 16 hours and EGFR location was assessed by subcellular fractionation. Lapatinib induced cytosolic translocation of EGFR in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 cells. Histone H1 and α-tubulin were used to test the purity of the nuclear and cytosolic fractions respectively. Quantification of EGFR levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Lapatinib interrupts the interaction between BRCA1 and EGFR in triple negative breast cancer cell lines.

<p>(A–B) Reciprocal immunoprecipitation with (A) EGFR and (B) BRCA1 was performed in (from left to right) MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells following 16 hours of vehicle or 1 µM lapatinib treatment. The levels of BRCA1 and EGFR in immunocomplexes were normalized to the amount of the reciprocal protein that was pulled down. (C–H) Quantification of EGFR and BRCA1 levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Combined EGFR and PARP inhibition delays the growth of orthotopic breast tumor xenografts in mice.

<p>MDA-MB-231 cells were injected into the mammary fat pads of mice. Once tumors were palpable, mice were treated with vehicle, 30 mg/kg/day lapatinib (b.i.d.), 100 mg/kg/day ABT-888 (b.i.d.), or combination of lapatinib and ABT-888. Treatment period was for 26 days and tumors were measured via caliper three times per week (n = 7, mean +/− SEM, ***p<0.001).</p

Treatment with lapatinib sequesters BRCA1 away from its nuclear repair substrates to the cytosol in triple negative breast cancer cells.

<p>(A–C) Cells were subjected to 1 µM lapatinib treatment for 16 hours and BRCA1 location was assessed by subcellular fractionation. Lapatinib induced cytosolic translocation of BRCA1 in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 cells. Histone H1 and α-tubulin were used to test the purity of the nuclear and cytosolic fractions respectively. Quantification of BRCA1 levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Kinase Gene Expression Profiling of Metastatic Clear Cell Renal Cell Carcinoma Tissue Identifies Potential New Therapeutic Targets - Fig 1

<p>Clustering of kinase genes showing A) normal tissue on the left (N, red), tumor in the middle (T, blue) and metastatic tumor tissue on the right (M, purple), and B) hierarchical clustering of all normal (N, red), tumor (T, blue), and metastatic (M, purple) tissues. Figure legend: X-axis represents the tissue sample, Y-axis represents the kinases. Yellow: up regulated kinases, Blue: down regulated kinases.</p